Differential subcellular distribution and transcriptional activity of sigmaE3, sigmaE4, and sigmaE3-4 isoforms of the rat estrogen receptor-alpha.

E3, E4, and E3-4 are naturally occurring estrogen receptor (ER) isoforms, generated through differential splicing of the ERalpha primary transcript and abundantly expressed in embryonic rat pituitary. Studies in COS cells transfected with full-length ERalpha or its three splice variants fused to green fluorescent protein (GFP), revealed a different subcellular localization for each isoform. In the absence of estradiol, full-length ERalpha-GFP was predominantly nuclear, and E3-GFP and E4-GFP were present both in cytoplasm and nucleus, whereas E3-4-GFP was predominantly cytoplasmic. Upon hormone treatment, a dramatic redistribution of full-length ERalpha-GFP and E3-GFP, from a diffuse to punctate pattern, occurred within the nucleus. In contrast, the distribution of E4-GFP and E3-4-GFP was unaffected. Nuclear fractionation studies showed that full-length ER-alpha and E3 displayed the same hormone-induced ability to tether to nuclear matrix, whereas nuclear E4 appeared to remain loosely associated to functional nuclear constituents. When cotransfected with an estrogen-inducible reporter plasmid (VIT-TK-CAT) in ER-negative (CHO k1) and ER-positive pituitary (GH4 C1) cells, E3-4 exhibited a very weak estrogen-dependent transactivation activity, whereas E3 had an inhibitory effect on full-length ER action. Conversely, E4 displayed estrogen-independent transcriptional activity in ER-negative cells, and in ER-positive cells, enhanced the estrogen-induced gene expression as efficiently as full-length ERalpha. In a gel mobility shift assay, phosphorylated E4 was able to form a specific complex with a consensus ERE, while E3 and E3-4 never did bind by themselves. The observed inhibitory action of E3 on estrogen-dependent transcription would rather involve protein-protein interactions such as formation of heterodimers with full-length ERalpha, as suggested by immunoprecipitation followed by Western blotting. These data suggest that E3 and E4 may play a physiologically relevant role as negative or constitutively positive modulators of transcription, in the developing rat pituitary.